Tick chitinase

ABSTRACT

A novel chitinase, a polynucleotide encoding the same, a vector and a transformant comprising the polynucleotide, an antibody against the chitinase, and a screening method for screening a substance capable of modifying the chitinase, are disclosed. According to the chitinase, polynucleotide, or vector, it is possible, for example, to exterminate ticks, or to treat or prevent tick-borne infections such as piroplasmosis, Q fever, or viral encephalitis.

TECHNICAL FIELD

This invention relates to a tick chitinase.

BACKGROUND ART

Ticks are the cause, directly or indirectly, of extensive damage toanimals or humans. Examples of the direct damage are pruritus orbleeding caused by biting or blood-sucking, or tick paralysis orallergic diseases caused by saliva secreted in blood-sucking orregurgitation of midgut contents. Examples of the indirect damage arevarious diseases in livestock, caused by viruses, rickettsiae,bacterium, spirochaeta, protozoa, nematoda, or the like. This damagecauses enormous losses at home and abroad, and threat of emerging andre-emerging zoonotic diseases by ticks is becoming a serious problem.

Under these circumstances, various methods to exterminate ticks are usedin many countries. Among these methods, the major one is the use ofagents such as organic phosphorus agents, carbamate agents, pyrethroidor macrolide antibiotics, or the like. However, in any agent, a drugresistance is established by using the agent successively or heavily,and thus many agents lose their miticidal activity. Further, when usingsuch agents, it is necessary to take side effects to animals intoconsideration. In addition, there is a problem of a remnant agent whichmay threaten the safety of foods and the environment, and people tend toavoid the use of such agents. Furthermore, the use of agents isapproaching limitation, with respect to the enormous development cost,in addition to the effectiveness thereof and an applicable area. Asdescribed above, it is considered difficult to prevent the parasitism ofticks to humans or animals, and the damage caused by ticks-borneinfection in the 21st century, by means of the use of agents.

In hematophagous arthropods including ticks, acquisition of protectiveimmune response against reinfection in a host against a viral orbacterial infection is known and has been confirmed in the laboratorystage [Fujisaki, Nat. Inst. Anim. Hlth. Quart. (Tokyo), 18, 27-38(1978)]. Due to the recent progress in gene recombination techniques,genes encoding protective antigens, enzymes related to metamorphosisspecific to hematophagous arthropods, or the like are being intensivelycloned in many countries, and an attempt to manufacture safe vaccineproteins or chemotherapeutic agents has been made.

However, such an agent in practical use is only that against Boophilusmicroplus, which was developed by Willadesen [Willadesen and Jogejan,Prasitology Today. 15, 258-262 (1999)]. There is now a search for avaccine against Haemaphysalis longicornis, which is widely distributedover Asian countries including Japan and the Eurasia continent andmediates zoonotic diseases such as piroplasmosis, Q fever, or viralencephalitis and thus the rapid development and practical application ofsuch a vaccine is greatly desired.

DISCLOSURE OF INVENTION

The present inventors have conducted intensive studies into obtaining anovel polypeptide useful as a candidate for a vaccine against ticks,particularly Haemaphysalis longicornis, and a polynucleotide encodingthe polypeptide and, as a result, found a novel chitinase and apolynucleotide encoding the same. Further, the present inventorsinoculated the chitinase into mice, to observe the induction of anantibody production, and confirmed that the chitinase is useful as atick vaccine. The present invention is based on the above findings.

The object of the present invention is to provide a novel chitinaseuseful as a tick vaccine, and a polynucleotide encoding the chitinase.

The object can be solved by a polypeptide of the present invention,i.e., (1) a polypeptide consisting of the amino acid sequence of SEQ IDNO: 2;

-   (2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2    and exhibiting a chitinase activity;-   (3) a polypeptide exhibiting a chitinase activity and comprising an    amino acid sequence in which one or plural amino acids are    substituted, deleted, and/or inserted at one or plural positions in    the amino acid sequence of SEQ ID NO: 2; or-   (4) a polypeptide comprising an amino acid sequence having a 60% or    more homology with the amino acid sequence of SEQ ID NO: 2, and    exhibiting a chitinase activity.

The present invention relates to a polynucleotide encoding thepolypeptide.

The present invention relates to a vector comprising the polynucleotide.

The present invention relates to a transformant comprising thepolynucleotide.

The present invention relates to a process for producing thepolypeptide, comprising the step of culturing the transformant.

The present invention relates to a medicament comprising the polypeptideor a fragment thereof, the polynucleotide, or the vector.

The present invention relates to a pharmaceutical composition comprisingthe polypeptide or a fragment thereof, the polynucleotide, or thevector, and a pharmaceutically or veterinary acceptable carrier ordiluent.

The present invention relates to a method for exterminating ticks,comprising administering to a subject in need thereof the polypeptide ora fragment thereof, the polynucleotide, or the vector in an amounteffective therefor.

The present invention relates to a method for treating or preventing atick-borne infection, comprising administering to a subject in needthereof the polypeptide or a fragment thereof, the polynucleotide, orthe vector in an amount effective therefor.

The present invention relates to an antibody or a fragment thereof,which binds to the polypeptide.

The present invention relates to a method for screening a substancecapable of modifying a chitinase activity of the polypeptide, comprisingthe steps of:

-   bringing the polypeptide into contact with a substance to be tested;    and-   analyzing the chitinase activity of the polypeptide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the result of SDS-polyacrylamide gel electrophoresis of arecombinant chitinase fusion protein.

FIG. 2 shows the result of electrophoresis of native (wild type)chitinase in a tick egg lysate by immunoblotting.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

[1] Polypeptide of the Present Invention

The polypeptides of the present invention includes

-   (1) a polypeptide consisting of the amino acid sequence of SEQ ID    NO: 2;-   (2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2    and exhibiting a chitinase activity;-   (3) a polypeptide comprising an amino acid sequence in which one or    plural amino acids are substituted, deleted, and/or inserted at one    or plural positions in the amino acid sequence of SEQ ID NO: 2, and    exhibiting a chitinase activity (hereinafter referred to as a    variation functionally equivalent); and-   (4) a polypeptide comprising an amino acid sequence having a 60% or    more homology with the amino acid sequence of SEQ ID NO: 2, and    exhibiting a chitinase activity (hereinafter referred to as a    homologous polypeptide).

The term “chitinase activity” as used herein means an enzyme activity inwhich chitin [poly(β-1,4-N-acetyl D-glucosamine)] is digested togenerate oligosaccharides and N-acetylglucosamine. Whether or not apolypeptide to be tested exhibits the chitinase activity may be easilyconfirmed, for example, by a known method for measuring the chitinaseactivity, in which the polypeptide to be tested is brought into contactwith a substrate of chitinase, and then the digestion and/or a degreethereof of the chitinase substrate is analyzed [for example, Johannes etal., Infect. Immun., 69, 4041-4047 (2001)]. The method is notparticularly limited, but is preferably confirmed by a method describedin Example 6.

More particularly, for example, a polypeptide to be tested is added to awell of an agarose gel containing an appropriate substrate of chitinase(for example, glycol chitin or chitin), and incubated for apredetermined period (for example, at 37° C. for 12 hours). The gel isstained with an appropriate dye [for example, Fluorescent Brightener 28(Sigma)] and observed under an ultraviolet ray. The portion in whichchitin is digested by chitinase does not react with the dye, and becomesblack. In this case, it may be judged that the polypeptide to be testedexhibits the chitinase activity. Conversely, when the chitinase reactiondoes not occur, the gel is brightened by the reaction with the dye. Inthis case, it may be judged that the polypeptide to be tested does notexhibit the chitinase activity.

The “polypeptide comprising the amino acid sequence of SEQ ID NO: 2 andexhibiting the chitinase activity” as the polypeptide of the presentinvention includes, for example,

-   a fusion polypeptide consisting of an amino acid sequence in which    an appropriate marker sequence or the like is added to the    N-terminus and/or the C-terminus of the amino acid sequence of SEQ    ID NO: 2, and exhibiting the chitinase activity; or-   a fusion polypeptide of the polypeptide consisting of the amino acid    sequence of SEQ ID NO: 2 and a partner for fusion, and exhibiting    the chitinase activity.

As the marker sequence, for example, a sequence for easily carrying outa confirmation of polypeptide expression, a confirmation ofintracellular localization thereof, a purification thereof, or the likemay be used. As the sequence, there may be mentioned, for example, aFLAG tag, a hexa-histidine tag, a hemagglutinin tag, a myc epitope, orthe like.

As the partner for fusion, there may be mentioned, for example, apolypeptide for purification [for example, glutathione S-transferase(GST) or a fragment thereof], a polypeptide for detection [for example,hemagglutinin or β-galactosidase α peptide (LacZ α), or a fragmentthereof], a polypeptide for expression (for example, a signal sequence),or the like.

In the above fusion polypeptide, an amino acid sequence which can bespecifically digested with a protease such as thrombin or factor Xa maybe optionally inserted between the polypeptide consisting of the aminoacid sequence of SEQ ID NO: 2 and the marker sequence or the partner forfusion.

The variation functionally equivalent of the present invention is notparticularly limited, so long as it is a polypeptide comprising an aminoacid sequence in which one or plural (preferably 1 to 10, morepreferably 1 to 7, most preferably 1 to 5) amino acids in total (forexample, one to several amino acids in total) are deleted, substituted,and/or inserted at one or plural positions in the amino acid sequence ofSEQ ID NO: 2, and exhibiting the chitinase activity. Further, an originof the variation functionally equivalent is not limited to Haemaphysalislongicornis.

The variation functionally equivalent of the present invention includesnot only Haemaphysalis longicornis variations of the polypeptideconsisting of the amino acid sequence of SEQ ID NO: 2, but alsovariations functionally equivalent derived from organisms other thanHaemaphysalis longicornis [for example, Ixodids (hard ticks) other thanHaemaphysalis longicornis, or Argasids (soft ticks)]. Further, itincludes polypeptides prepared using polynucleotides obtained byartificially modifying their amino acid sequences encoded thereby bygenetic engineering techniques, on the basis of polynucleotides encodingthese native polypeptides (i.e., Haemaphysalis longicornis variations orvariations functionally equivalent derived from organisms other thanHaemaphysalis longicornis), or on the basis of polynucleotides encodingthe amino acid sequence of SEQ ID NO: 2. The term “variation” as usedherein means an individual difference between the same polypeptides inthe same species or a difference between homologous polypeptides inseveral species.

Haemaphysalis longicornis variations of the polypeptide consisting ofthe amino acid sequence of SEQ ID NO: 2, or variations functionallyequivalent derived from organisms other than Haemaphysalis longicornismay be obtained by those skilled in the art in accordance with theinformation of a base sequence (for example, the base sequence of the571st to 3360th bases in the base sequence of SEQ ID NO: 1) of apolynucleotide encoding the polypeptide consisting of the amino acidsequence of SEQ ID NO: 2. In this connection, genetic engineeringtechniques may be generally performed in accordance with known methods(for example, Sambrook et al., “Molecular Cloning, A Laboratory Manual”,Cold Spring Harbor Laboratory Press, 1989), unless otherwise specified.

For example, an appropriate probe or appropriate primers are designed inaccordance with the information of a base sequence of a polynucleotideencoding the polypeptide consisting of the amino acid sequence of SEQ IDNO: 2. A polymerase chain reaction (PCR) method (Saiki, R. K. et al.,Science, 239, 487-491, 1988) or a hybridization method is carried outusing a sample (for example, total RNA or an mRNA fraction, a cDNAlibrary, or a phage library) derived prepared from an organism [forexample, Ixodids (hard ticks) other than Haemaphysalis longicornis, orArgasids (soft ticks)] of interest and the primers or the probe toobtain a polynucleotide encoding the polypeptide. A desired polypeptidemay be obtained by expressing the resulting polynucleotide in anappropriate expression system and confirming that the expressedpolypeptide exhibits the chitinase activity by, for example, the methoddescribed in Example 6.

Further, the polypeptide artificially modified by genetic engineeringtechniques may be obtained by, for example, the following procedure. Agene encoding the polypeptide is obtained by a conventional method suchas site-specific directed mutagenesis (Mark, D. F. et al., Proc. Natl.Acad. Sci. USA, 81, 5662-5666, 1984). A desired polypeptide may beobtained by expressing the resulting polynucleotide in an appropriateexpression system and confirming that the expressed polypeptide exhibitsthe chitinase activity by, for example, the method described in Example6.

The homologous polypeptide of the present invention is not particularlylimited, so long as it is a polypeptide having an amino acid sequencehaving a 60% or more homology with the amino acid sequence of SEQ ID NO:2, and exhibiting the chitinase activity. The homologous polypeptide ofthe present invention may have an amino acid sequence having preferablya 70% or more homology, more preferably a 80% or more homology, morepreferably a 90% or more homology, more preferably a 95% or morehomology, most preferably a 98% or more homology, with respect to theamino acid sequence of SEQ ID NO: 2. The term “homology” as used hereinmeans a value obtained by a Clustal program (Higgins and Sharp, Gene,73, 237-244, 1988; and Thompson et al., Nucleic Acid Res., 22,4673-4680, 1994) in accordance with a default parameter.

The above-mentioned novel polypeptide of the present invention may bemanufactured by various known methods, for example, known geneticengineering techniques using the polynucleotide of the present inventionwhich encodes the polypeptide of the present invention. Moreparticularly, the polypeptide of the present invention may be preparedby culturing the transformant of the present invention described below(i.e., the transformant comprising the polynucleotide of the presentinvention) under conditions in which the novel polypeptide of thepresent invention can be expressed, and then separating and purifyingthe desired polypeptide from the resulting culture in accordance with aconventional method for a polypeptide separation and purification. Asthe separation and purification method, there may be mentioned, forexample, salting-out with ammonium sulfate, an ion exchange columnchromatography using ion exchange cellulose, a molecular sieve columnchromatography using molecular sieve gel, an affinity columnchromatography using protein A agarose, dialysis, lyophilization, or thelike.

The present invention includes a fragment of the polypeptide of thepresent invention. The fragment of the present invention is useful as anactive ingredient for the medicament of the present invention or as anantigen for preparing the antibody of the present invention.

[2] Polynucleotide of the Present Invention

The polynucleotide of the present invention is not particularly limited,so long as it encodes the polypeptide of the present invention. As thepolynucleotide of the present invention, there may be mentioned, forexample, a polynucleotide consisting of the 571st to 3360th bases in thebase sequence of the SEQ ID NO: 1. In this connection, the term“polynucleotide” as used herein includes both DNA and RNA.

The present invention includes a polynucleotide comprising a basesequence which can hybridize with the polynucleotide of the presentinvention, preferably a polynucleotide consisting of a base sequencewhich can hybridize with the polynucleotide of the present invention.The base sequence capable of hybridizing with the polynucleotide of thepresent invention is preferably a base sequence complementary to thebase sequence (or a partial sequence thereof) of the polynucleotide ofthe present invention, more preferably a base sequence complementary tothe base sequence (or a partial sequence thereof) consisting of the571st to 3360th bases in the base sequence of the SEQ ID NO: 1.

[3] Vector and Transformant of the Present Invention

The vector of the present invention is not particularly limited, so longas it comprises the polynucleotide of the present invention. As thevector, there may be mentioned, for example, a vector obtained byintroducing the polynucleotide of the present invention into a knownexpression vector appropriately selected in accordance with a host cellto be used.

The transformant of the present invention is not particularly limited,so long as it comprises the polynucleotide of the present invention. Thetransformant of the present invention may be, for example, a cell inwhich the polynucleotide is integrated into a chromosome of a host cell,or a transformant containing the polynucleotide as a vector comprisingpolynucleotide. Further, the transformant of the present invention maybe a transformant expressing the polypeptide of the present invention,or a transformant not expressing the polypeptide of the presentinvention. The transformant of the present invention may be obtained by,for example, transfecting a desired host cell with the vector of thepresent invention or the polynucleotide of the present invention per se.

The host cell may be, for example, a known microorganism usually used,for example, an Escherichia coli or yeast (Saccharomyces cerevisiae), ora known cultivated cell, such as an animal cell, such as a CHO cell, anHEK-293 cell, or a COS cell, or an insect cell such as a BmN4 cell.

The known expression vector may be, for example, pUC, pTV, pGEX, pKK, orpTrcHis for an Escherichia coli; pEMBLY or pYES2 for the yeast; pcDNA3or pMAMneo for the CHO cell; pcDNA3 for the HEK-293 cell; pcDNA3 for theCOS cell; a vector (such as pBK283) containing a polyhedrin promoter ofa silkworm nucleopolyhederovirus (BmNPV) for the BmN4 cell. Further, theexpression vector includes a virus vector which can be used as a vectorfor a gene therapy, such as retrovirus, adenovirus, or Sendai virus.

[4] Medicament of the Present Invention

The medicament of the present invention (preferably a tick vaccine)comprises, as a active ingredient, the polypeptide of the presentinvention or a fragment thereof, the polynucleotide of the presentinvention, or the vector of the present invention. In the presentinvention, the polypeptide of the present invention or a fragmentthereof, the polynucleotide of the present invention, or the vector ofthe present invention can be orally or parenterally administered alone,or preferably together with a pharmaceutically or veterinarilyacceptable carrier or diluent, to an animal (preferably a mammal,particularly a human) in need of an extermination of ticks.

When the active ingredient in the medicament of the present invention(i.e., the polypeptide of the present invention or a fragment thereof,the polynucleotide of the present invention, or the vector of thepresent invention) is administered to an animal as a tick vaccine, anantibody production may be induced and then ticks may be terminated byprotective immune response against reinfection in the host animal.Further, as a result, it is possible to treat or prevent tick-borneinfections such as piroplasmosis, Q fever, or viral encephalitis.

In other words, the pharmaceutical composition (preferablypharmaceutical composition for exterminating ticks or pharmaceuticalcomposition for treating or preventing a tick-borne infection) of thepresent invention comprises the polypeptide of the present invention ora fragment thereof, the polynucleotide of the present invention, or thevector of the present invention as the active ingredient, and apharmaceutically or veterinary acceptable carrier or diluent. The activeingredient in the present invention (i.e., the polypeptide of thepresent invention or a fragment thereof, the polynucleotide of thepresent invention, or the vector of the present invention) can be usedin the manufacture of the above medicament (preferably medicament forexterminating ticks or medicament for treating or preventing atick-borne infection).

When the medicament of the present invention is used as a tick vaccine,the fragment of the polypeptide of the present invention is notparticularly limited, so long as the fragment administered to a subjectcan induce immunity thereagainst. The fragment can be appropriatelyselected by those skilled in the art.

The medicament (particularly the tick vaccine) of the present inventioncan be used, for example, by mixing the polypeptide of the presentinvention with an adjuvant or the like and inoculating the resultingmixture into an animal (for example, livestock) at an appropriateinterval as a tick vaccine. Further, it can be used by dissolving orsuspending the polypeptide of the present invention directly in anappropriate solvent, or by enclosing it in liposomes or integrating aDNA encoding it in an appropriate vector. Furthermore, it can be used inan appropriate formulation such as injections, tablets, capsules, eyedrops, creams, suppositories, sprays, poultices, or the like, optionallyby adding a pharmaceutical acceptable carrier to the polypeptide of thepresent invention.

As the pharmaceutical acceptable carrier, well-known solvents, bases,stabilizing agents, antiseptics, solubilizing agents, fillers, buffers,and the like may be used. When the polypeptide of the present inventioncontained in the medicament of the present invention is used in theabove formulation, the administration method and the dose may bedetermined in accordance with, for example, the age or sex of eachsubject, or the kind or degree of each disease.

The oral administration includes a sublingual administration. As theparenteral administration, for example, inhalation, percutaneousadministration, ophthalmic administration, vaginal administration,intra-articular administration, rectal administration, intra-arterialadministration, intravenous administration, local administration,intramuscular administration, subcutaneous administration,intraperitoneal administration, or the like may be selected.

[5] Antibody and the Fragment thereof of the Present Invention

An antibody, such as a polyclonal antibody or a monoclonal antibody,which reacts with the polypeptide of the present invention may beobtained by directly administering the polypeptide of the presentinvention or a fragment thereof to various animals. Alternatively, itmay be obtained by a DNA vaccine method (Raz, E. et al., Proc. Natl.Acad. Sci. USA, 91, 9519-9523, 1994; or Donnelly, J. J. et al., J.Infect. Dis., 173, 314-320, 1996), using a plasmid into which apolynucleotide encoding the polypeptide of the present invention isinserted.

The polyclonal antibody may be produced from a serum or eggs of ananimal such as a rabbit, a rat, a goat, or a chicken, in which theanimal is immunized and sensitized by the polypeptide of the presentinvention or a fragment thereof emulsified in an appropriate adjuvant(for example, Freund's complete adjuvant) by intraperitoneal,subcutaneous, or intravenous administration. The polyclonal antibody maybe separated and purified from the resulting serum or eggs in accordancewith conventional methods for polypeptide isolation and purification.Examples of the separation and purification methods include, forexample, centrifugal separation, dialysis, salting-out with ammoniumsulfate, or a chromatographic technique using such as DEAE-cellulose,hydroxyapatite, protein A agarose, and the like.

The monoclonal antibody may be easily produced by those skilled in theart, according to, for example, a cell fusion method of Kohler andMilstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).

A mouse is immunized intraperitoneally, subcutaneously, or intravenouslyseveral times at an interval of a few weeks by a repeated inoculation ofemulsions in which the polypeptide of the present invention or afragment thereof is emulsified into a suitable adjuvant such as Freund'scomplete adjuvant. Spleen cells are removed after the finalimmunization, and then fused with myeloma cells to prepare hybridomas.

As a myeloma cell for obtaining a hybridoma, a myeloma cell having amarker such as a deficiency in hypoxanthine-guaninephosphoribosyltransferase or thymidine kinase (for example, mousemyeloma cell line P3X63Ag8.U1) may be used. As a fusing agent,polyethylene glycol may be used. As a medium for preparation ofhybridomas, for example, a commonly used medium such as an Eagle'sminimum essential medium, a Dulbecco's modified minimum essentialmedium, or an RPMI-1640 medium may be used by adding properly 10 to 30%of a fetal bovine serum. The fused strains may be selected by a HATselection method. A culture supernatant of the hybridomas is screened bya well-known method such as an ELISA method or an immunohistologicalmethod, to select hybridoma clones secreting the antibody of interest.The monoclonality of the selected hybridoma is guaranteed by repeatingsubcloning by a limiting dilution method. Antibodies in an amount whichmay be purified are produced by culturing the resulting hybridomas in amedium for 2 to 4 days, or in the peritoneal cavity of apristane-pretreated BALB/c strain mouse for 10 to 20 days.

The resulting monoclonal antibodies in the culture supernatant or theascites may be separated and purified by conventional polypeptideisolation and purification methods. Examples of the separation andpurification methods include, for example, centrifugal separation,dialysis, salting-out with ammonium sulfate, or chromatographictechnique using such as DEAE-cellulose, hydroxyapatite, protein Aagarose, and the like.

Further, the monoclonal antibodies or the antibody fragments containinga part thereof may be produced by inserting the whole or a part of agene encoding the monoclonal antibody into an expression vector andintroducing the resulting expression vector into appropriate host cells(such as E. coli, yeast, or animal cells).

Antibody fragments comprising an active part of the antibody such asF(ab′)₂, Fab, Fab′, or Fv may be obtained by a conventional method, forexample, by digesting the separated and purified antibodies (includingpolyclonal antibodies and monoclonal antibodies) with a protease such aspepsin, papain, and the like, and separating and purifying the resultingfragments by standard polypeptide isolation and purification methods.

Further, an antibody which reacts to the polypeptide of the presentinvention may be obtained in a form of single chain Fv or Fab inaccordance with a method of Clackson et al. or a method of Zebedee etal. (Clackson, T. et al., Nature, 352, 624-628, 1991; or Zebedee, S. etal., Proc. Natl. Acad. Sci. USA, 89, 3175-3179, 1992). Furthermore, ahumanized antibody may be obtained by immunizing a transgenic mouse inwhich mouse antibody genes are substituted with human antibody genes(Lonberg, N. et al., Nature, 368, 856-859, 1994).

[6] Screening Method of the Present Invention

It is possible to determine whether or not a substance to be testedmodifies (for example, suppresses or promotes) the chitinase activity ofthe polypeptide according to the present invention, using thepolypeptide of the present invention.

Substances to be tested to which may be applied the screening method ofthe present invention are not particularly limited, but there may bementioned, for example, various known compounds (including peptides)registered in chemical files, compounds obtained by combinatorialchemistry techniques (Terrett, N. K. et al., Tetrahedron, 51, 8135-8137,1995), or random peptides prepared by employing a phage display method(Felici, F. et al., J. Mol. Biol., 222, 301-310, 1991) or the like. Inaddition, culture supernatants of microorganisms, natural componentsderived from plants or marine organisms, or animal tissue extracts maybe used as the test substances for screening. Further, compounds(including peptides) obtained by chemically or biologically modifyingcompounds (including peptides) selected by the screening method of thepresent invention may be used.

The screening method of the present invention may be carried out by amethod similar to the above-mentioned method for confirming thechitinase activity, except that the polypeptide of the presentinvention, a substrate of chitinase, and the test substance are broughtinto contact with each other instead of bringing the test polypeptideinto contact with a substrate of chitinase.

Namely, in the screening method of the present invention, it isconfirmed whether or not the test substance modifies the chitinaseactivity of the polypeptide of the present invention by bringing intocontact the polypeptide of the present invention, a substrate ofchitinase, and the test substance, and then analyzing whether or not thesubstrate of chitinase is digested (or a degree of the digestion) by thechitinase activity of the polypeptide of the present invention in thepresence of the test substance. When the substrate of chitinase is notdegraded by the chitinase activity of the polypeptide of the presentinvention, or the degree of the digestion is decreased, it is possibleto confirm that the test substance suppresses the chitinase activity ofthe polypeptide of the present invention. Alternatively, when the degreeof the digestion of the substrate of chitinase by the chitinase activityof the polypeptide of the present invention is increased, it is possibleto confirm that the test substance promotes the chitinase activity ofthe polypeptide of the present invention.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples. The procedures described inthe following Examples were performed in accordance with varioustechniques commonly used in molecular biology, acarology,arthropodology, immunology, or biochemistry, described in, for example,Sambrook et al., “Molecular Cloning, A Laboratory Manual”, Cold SpringHarbor Laboratory Press, 1989 or similar books. As a software foranalyzing DNA, MacVector™ (Oxford Molecular) was used.

Example 1 Isolation of a Gene Encoding a Novel Tick Chitinase

Total RNA was extracted from eggs of a Haemaphysalis longicornis Okayamastrain [Fujisaki et al., Nat. Inst. Anim. Hlth Quart. (Tokyo), 16,122-128 (1976)] by an Acid Guanidinium-phenol-chloroform method[Chomczynski et al., Anal. Biochem., 162, 156-159 (1987)]. From theresulting total RNA, poly A⁺ RNA was purified using an mRNA isolationkit [Oligotex-dT30 (Super), code W9021B; Takara] in accordance with aprotocol attached to the kit.

The following procedures, i.e., construction of a cDNA library,screening, and insertion into plasmid of a cDNA clone (in vivo Excision)were performed using commercially available reagent kits (Stratagen) inaccordance with protocols attached thereto.

More particularly, cDNA was synthesized using 5 μg of tick mRNA as atemplate and a cDNA synthesis kit (ZAP-cDNA Synthesis Kit, Cat. No.200401-5; Stratagen). The resulting cDNA was fractionated by a sizefractionation with a Sepharose CL-2B gel column, inserted into a vector(Uni-ZAP XR Vector, Cat. No. 237211; Stratagen), and packaged using apackaging reagent (GigapackIII Gold packaging extract; Stratagen) .Escherichia coli (E. coli XL1-Blue MRF′ strain) was transfected with thepackaged product to obtain a library containing approximately 500,000cDNA clones.

The cDNA library was plaque-screened using a polymerase chain reaction(PCR) method, to obtain four overlapping positive clones. Moreparticularly, a fragment of a tick chitinase gene obtained by PCR, as aprobe, was reacted with the cDNA library. A positive colony was observedas a black dot. Screening was performed by confirming the black dots. Inthis connection, the PCR was carried out using two primers having thebase sequences of SEQ ID NOS: 7 and 8 designed from the amino acidsequences of SEQ ID NOS: 5 and 6, respectively. In the PCR, 50 μL of areaction liquid [template DNA 1 μg, 0.1 μmol/L primer, 10 mmol/LTris-HCl (pH8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, and 2.5U Taq Gold DNApolymerase (Part No. N808-0244; Perkin Elmer)] was used, and a cyclecomposed of treatments at 94° C. for 1 minute, 50° C. for 1 minute, and72° C. for 2 minutes was repeated 40 times.

These positive clones were inserted into plasmid (i.e., converted into apBluescript) by an in vivo Excision method.

Each plasmid containing a cDNA fragment was purified using a plasmidpurification kit (Cat no. 12125; Qiagen), and then PCR was carried outusing a sequencing kit (Dye Primer Cycle Sequencing Kit, Part No.4303153; Perkin Elmer) in accordance with a protocol attached to thekit. Each resulting PCR product was analyzed with a DNA sequencer (ABIPRISM 3100 Genetic Analyzer; Perkin Elmer) to determine a base sequenceof each cDNA fragment.

As a result, it was found that all four clones were derived from asingle gene. The longest clone was used in the following analyses.

The full length of the cDNA was 6439 bp, and the base sequence thereofwas that of SEQ ID NO: 1. It was confirmed that the base sequencecontains an open reading frame consisting of 2790 bp (a base sequenceconsisting of the 571st to 3360th bases in the base sequence of SEQ IDNO: 1). The amino acid sequence of a protein deduced from the openreading frame was the amino acid sequence of SEQ ID NO: 2 consisting of929 amino acid residues, and the deduced molecular weight was 104 kDa.

The homology search of the deduced amino acid sequence was carried outby a BLAST method (Basic local alignment search tool; Altschul, S. F. etal., J. Mol. Biol., 215, 403-410, 1990; obtained from the NationalCenter for Biotechnology Information). As a result, it was confirmedthat the amino acid sequence had a high homology with known chitinaseproteins derived from other organisms. For example, it had anapproximately 30% homology with yellow fever mosquito (Aedes Egypt)chitinase [Insect Mol. Biol., 7(3), 233-239 (1998)].

Example 2 Construction of Vector for Expressing Tick Chitinase FusionProtein

For cloning the ORF region of the tick chitinase gene, a DNAamplification was carried out by PCR using the cDNA clone obtained inExample 1 as a template, and a sense primer (consisting of the basesequence of SEQ ID NO: 3 and containing the EcoRI recognition site“gaattc” at the 5′ terminus) and an antisense primer (consisting of thebase sequence of SEQ ID NO: 4 and containing the XhoI recognition site“ctcgag” at the 5′ terminus). In the PCR, 50 μL of a reaction liquid[template DNA 1 μg, 0.1 μmol/L primer, 10 mmol/L Tris-HCl (pH8.3), 50mmol/L KCl, 1.5 mmol/L MgCl₂, and 2.5U Taq Gold DNA polymerase (Part No.N808-0244; Perkin Elmer)] was used, and a cycle composed of treatmentsat 94° C. for 1 minute, 50° C. for 1 minute, and 72° C. for 2 minuteswas repeated 40 times.

The PCR product was treated with phenol/chloroform, collected by anethanol precipitation method, and dissolved in distilled water. Theresulting DNA solution was digested with the restriction enzyme EcoRI,and then the DNA fragment was separated by electrophoresis, purifiedusing a DNA purification kit (Cat no. 1001-400; Biotechnologies), andcollected in distilled water.

Meanwhile, a vector pGEMEX-4T-3 (Product no. 27-4583; Pharmacia Biotech)for expression in Escherichia coli was digested with the restrictionenzyme EcoRI, dephosphorylated with alkaline phosphatase, and purifiedin the manner similar to that used in the purification of the PCRproduct.

The purified PCR product and vector were reacted using a DNA ligationkit (Cat no. 6022; Takara) in accordance with a protocol attached to thekit. Escherichia coli DH5α was transformed with the ligation reactionproduct, and then recombinant clones in which the chitinase ORF fragmentwas inserted in the same direction as that of glutathione S-transferase(GST) in the vector were selected. A recombinant plasmid was purifiedusing a plasmid purification kit (Cat no. 12125; Qiagen).

Example 3 Expression of Tick Chitinase Recombinant Protein inEscherichia coli

Escherichia coli JM109 (DE3) (Promega) was transformed with therecombinant plasmid prepared in Example 2, and then the transformantswere cultured at 37° C. in an LB medium containing ampicillin. WhenOD_(600nm) of the culture became 0.3˜0.5, isopropyl-thio-galactoside(IPTG) was added to the culture so that the final concentration became0.5 mmol/L, and then the transformants were further cultured at 37° C.for 4 hours.

The expression of tick chitinase recombinant protein was confirmed bycarrying out 10% sodium dodecyl sulfate(SDS)-polyacrylamide gelelectrophoresis [Laemmli et al., Nature, 227, 680-685 (1970)] followedby Coomassie staining.

As a result, the expression of the recombinant protein having amolecular weight of approximately 130 kDa was observed, and it wasconfirmed that the recombinant protein was a fusion protein of a GSTleader protein (26 kDa) and the tick chitinase protein (104 kDa).

Example 4 Purification of Tick Chitinase Recombinant Protein andPreparation of Antiserum

The recombinant chitinase fusion protein expressed in Escherichia coliby the method described in Example 3 was purified in accordance with aprotocol attached to a commercially available kit (Bulk GST PurificationModule; Amersham Bioscience).

The result of electrophoresis of the purified recombinant chitinasefusion protein is shown in FIG. 1. In this connection, theelectrophoresis and staining were performed in the manner similar tothat described in Example 3. In FIG. 1, lane 1 is the result ofmolecular weight markers, lane 2 is the result of the purifiedrecombinant chitinase fusion protein, and lane 3 is the result of thepurified GST protein. The arrow at the right side of lane 3 indicatesthe recombinant chitinase fusion protein, and the numbers at the rightside of lane 3 mean the molecular weights of the recombinant chitinasefusion protein (130 kDa), and the tick chitinase protein (104 kDa) andthe GST leader protein (26 kDa) which are composed of the recombinantchitinase fusion protein.

An emulsion was prepared by mixing 200 μL of a solution containing 100μg of the purified recombinant chitinase fusion protein with 200 μL of acomplete Freund's adjuvant (Adjuvant Complete Freund; Difco). Theemulsion was intraperitoneally inoculated into a 8-week-old femaleBALB/c mouse. After 2 and 4 weeks from the intraperitoneal inoculation,100 μg of the recombinant chitinase fusion protein was mixed with anincomplete Freund's adjuvant (Difco), and each booster inoculation wascarried out. After 2 weeks from the final inoculation, blood wascollected and the resulting serum was kept at −20° C.

Example 5 Identification of Native (Wild Type) Chitinase byImmunoblotting

The wild type chitinase protein was identified by immunoblotting [Towbinet al., Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979)] using theanti-recombinant chitinase fusion protein mouse serum obtained inExample 4. As samples, a lysate of tick eggs, i.e., a supernatantprepared in accordance with a method described in You et al., InsectBiochem. Mol. Biol., 32, 67-73 (2000), was used. More particularly, thelasate was prepared by homogenizing tick eggs in 20 mmol/L Tris-HCl andcentrifuging the homogenate at 1000 rpm for 30 minutes.

The result is shown in FIG. 2. In FIG. 2, lane 1 is the result when theanti-recombinant chitinase fusion protein immune serum was used, andlane 2 is the result when the anti-GST protein immune serum was used(negative control). The arrow at the right side of lane 2 indicates thewild type chitinase protein, and the numbers at the right side of lane 2mean molecular weights.

As shown in lane 1 in FIG. 2, the specific band of the wild typechitinase protein (114 kDa) was detected in the egg lysate. The measuredmolecular weight was approximately 10 kDa higher than the molecularweight (104 kDa) deduced from the amino acid sequence of SEQ ID NO: 2.The difference seems to be due to glycosylation.

Example 6 Confirmation of Chitinase Activity in Recombinant ChitinaseFusion Protein

In this Example, the chitinase activity in the recombinant chitinasefusion protein was examined on a 1% agarose gel containing 0.01% glycolchitin, in accordance with a known method for measuring the chitinaseactivity [Johannes et al., Infect. Immun., 69, 4041-4047 (2001)]. Forcomparison, the chitinase activity in Serratia marcescens chitinase(Sigma ) or β-galactosidase was examined.

More particularly, 10 μL of each proten solution obtained by dissolvingeach protein in PBS (phosphate buffer) was added to each well in theagarose gel. After an incubation at 37° C. for 12 hours, the gel wasstained in a 0.01% staining liquid (Fluorescent Brightener28; Sigma),washed with distilled water, and observed under an ultraviolet ray.

The recombinant chitinase fusion protein or Serratia marcescenschitinase reacted with chitin more strongly than did the β-galactosidaseor PBS (control). As a result, it was confirmed that the recombinantchitinase fusion protein or Serratia marcescens chitinase exhibits aproperty of digesting chitin.

INDUSTRIAL APPLICABILITY

According to the polypeptide, polynucleotide, vector, transformant, andantibody of the present invention, the medicament of the presentinvention, particularly a tick vaccine, can be provided.

Further, according to the medicament of the present invention,particularly a tick vaccine, it is possible, for example, to exterminateticks, or to treat or prevent tick-borne infections such aspiroplasmosis, Q fever, or viral encephalitis.

Free Text in Sequence Listing

Each of the base sequences of SEQ ID NOS: 3, 4, 7, and 8 in the SequenceListing is an artificially synthesized primer sequence.

In the base sequence of SEQ ID NO: 8 in the Sequence Listing, thealphabet “n” means A (i.e., adenine), C (i.e., cytosine), G (i.e.,guanine), or T (i.e., thymine).

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2; (2) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2and exhibiting a chitinase activity; (3) a polypeptide exhibiting achitinase activity and comprising an amino acid sequence in which one orplural amino acids are substituted, deleted, and/or inserted at one orplural positions in the amino acid sequence of SEQ ID NO: 2; or (4) apolypeptide comprising an amino acid sequence having a 60% or morehomology with the amino acid sequence of SEQ ID NO: 2, and exhibiting achitinase activity.
 2. A polynucleotide encoding the polypeptideaccording to claims
 1. 3. A vector comprising the polynucleotideaccording to claim
 2. 4. A transformant comprising the polynucleotideaccording to claim
 2. 5. A process for producing the polypeptideaccording to claim 1, comprising the step of culturing the transformantaccording to claim
 4. 6. A medicament comprising the polypeptideaccording to claim 1 or a fragment thereof, the polynucleotide accordingto claim 2, or the vector according to claim
 3. 7. The medicamentaccording to claim 6, which is a tick vaccine.
 8. A pharmaceuticalcomposition comprising the polypeptide according to claim 1 or afragment thereof, the polynucleotide according to claim 2, or the vectoraccording to claim 3, and a pharmaceutically or veterinary acceptablecarrier or diluent.
 9. A method for exterminating ticks, comprisingadministering to a subject in need thereof the polypeptide according toclaim 1 or a fragment thereof, the polynucleotide according to claim 2,or the vector according to claim 3 in an amount effective therefor. 10.A method for treating or preventing a tick-borne infection, comprisingadministering to a subject in need thereof the polypeptide according toclaim 1 or a fragment thereof, the polynucleotide according to claim 2,or the vector according to claim 3 in an amount effective therefor. 11.An antibody or a fragment thereof, which binds to the polypeptideaccording to claim
 1. 12. A method for screening a substance capable ofmodifying a chitinase activity of the polypeptide according to claim 1,comprising the steps of: bringing the polypeptide into contact with asubstance to be tested; and analyzing the chitinase activity of thepolypeptide.